Microcarriers provide an alternative for large-scale cell culture. Microcarriers are typically stirred in cell culture media and provide a very large surface to area ratio for cell growth. Microcarriers can provide substantially higher cell yields per culture volume relative to conventional equipment.
For anchorage-dependent cells, microcarriers having positively charged surfaces have been shown to provide excellent adhesion and growth. However, cell harvesting may be difficult in proportion to the robustness of the adhesion and generally requires harsh protease treatment that may have deleterious effects on cells. In addition, such microcarriers can suffer from loss of cells and lack of reproducibility in stirred culture. Microcarrier toxicity and nutrient absorption have been identified as causes of these difficulties. Moreover, to achieve good cell attachment and proliferation charge density should be optimized. These problems have been partially overcome by coating the microcarriers, treating with a sequence of chemical or physical steps to reduce the charge, adding carboxymethyl cellulose, or soaking the beads in serum. While successful, such pretreatment of the carrier is time consuming introduces variability.
Some microcarriers have been designed to enable appropriate adhesion without extensive tuning of surface charges density. For example, microcarriers have been prepared to present specific polypeptide sequences at the surface, which polypeptides are configured to provide specific interaction with adhesion receptor of the cells. Examples of such microcarriers include gelatin or collagen linked to dextran beads or to polystyrene beads. While having various advantages, such microcarriers are made of animal derived materials and are not suitable for culturing cells dedicated to cell therapies due to the risk of xenogenic contamination through, for example, pathogen proteins or viruses.
To solve this issue, recombinant proteins or polypeptides have been synthesized and coated onto microcarriers. While such microcarries have the advantage of being free of animal derived components, they may have several drawbacks. For example, some microcarriers have been shown not only to bind cells, but also to activate cells, which means that the cell surface and cytoskeletal proteins have rearranged and specific genes have been induced to result in spreading and proliferation. Such activation may not be desirable when culturing cells, such as pluripotent stem cells. In addition, the level of cell adhesion with some of these beads may not be sufficiently high for robust culture of some anchorage-dependent cells, particularly when serum-free, chemically-defined media are used. When serum-free media are used, the media do not provide adhesion proteins, which can bind to the microcarrier surface and thus facilitate binding of cells. The absence of serum especially presents problems when using cells that produce little extracellular matrix, such as certain stem cells, including embryonic stem cells.
A good number of positively charged microcarriers, some of which contain associated protein or recombinant polypeptide, are available for use in culturing anchorage-dependent cells. These microcarriers have proven effective for culturing many cells under many conditions. However, they do suffer from the drawbacks described above.